Final project –Final project – Computational Computational

BiologyBiologyIdentifying Identifying

transcription transcription factories not factories not

involved in pre-involved in pre-mRNA splicingmRNA splicing

ניסים נעיםמגישים: עדי פוטוק

מר יהודה בהנחיית:ברודי

ד"ר ירון שב-טל

IntroductionIntroductionThere is evidence of transcription factoriesThere is evidence of transcription factorieswhich contain accumulations of RNA polymerase II.which contain accumulations of RNA polymerase II.Genes are moving towards the factories in order to beGenes are moving towards the factories in order to betranscribed.transcribed.

Splicing is a co-transcriptional modification of an mRNA,Splicing is a co-transcriptional modification of an mRNA,in which introns are removed and exons are joined.in which introns are removed and exons are joined.

U1 and U2 are parts of the spliceosome machinery.U1 and U2 are parts of the spliceosome machinery.

IntroductionIntroduction

http://www.mpibpc.mpg.de/groups/pr/PR/2008/08_10_eng_RN/

Identification and classification of transcription factories, and categorizing the factories by their probability to undergo splicing.

Project goalProject goal

RNA FISHRNA FISH

ImmunofluorescenceImmunofluorescence

Using a labeled oligonucleotide Using a labeled oligonucleotide probe to detect a specificprobe to detect a specificmRNA of interest.mRNA of interest.

Using a fluorescent labeledUsing a fluorescent labeledantibody to detect U1snRNA,antibody to detect U1snRNA,U1snRNA and RNA polymerase II.U1snRNA and RNA polymerase II.

Fluorescence In-Situ HybridizationFluorescence In-Situ Hybridization

mRNA

The cells are illuminated with lightThe cells are illuminated with light

of a certain wavelength and emitof a certain wavelength and emit

light of a different wavelengthlight of a different wavelength..

This technique is used to acquire 3DThis technique is used to acquire 3D

images of a specimen. Each imageimages of a specimen. Each image

is composed of several 2D layers.is composed of several 2D layers.

Wide-Field MicroscopyWide-Field Microscopy

ProblemProblem: The light emitted from the fluorescent: The light emitted from the fluorescentmolecules disperses, as the layer gets farther awaymolecules disperses, as the layer gets farther awayfrom the molecules.from the molecules.

SolutionSolution: this method is used to focus the light back: this method is used to focus the light backto its original source, in order to create an image moreto its original source, in order to create an image moresimilar to the original image.similar to the original image.

DeconvolutionDeconvolution

DeconvolutionDeconvolution

Before After

Image analysisImage analysis

IMARISIMARIS – – Tool for analyzing images Tool for analyzing images Wide graphical abilities.Wide graphical abilities. Embedded link to MATLAB programs.Embedded link to MATLAB programs.

Gene constructs: e1 and e3Gene constructs: e1 and e3

• e1 gene:e1 gene:

no splicingno splicing

• e3 gene:e3 gene:

undergoes splicingundergoes splicing

Splicing is co-transcriptional

sn

RN

AU

1

snRNAU2 snRNAU1

snRNAU2

MS2

e3

e1

MS2U1 snRNAU2 snRNA

Splicing factors can be identified at the site Splicing factors can be identified at the site of transcriptionof transcription

MS2U1 snRNAU2 snRNA

Transcription site

Nucleoplasm

Transcription site

Nucleoplasm

U1

snR

NA

U1

snR

NA

U2 snRNA

U2 snRNA

Splicing is co-transcriptional

sn

RN

AU

1

Splicing factors can be identified at the site Splicing factors can be identified at the site of transcriptionof transcription

RNA pol II ImmunofluorescenceRNA pol II Immunofluorescence

Nucleoplasm

Nucleolus

Transcription factory

Cytoplasm

Step I – Identify FactoriesStep I – Identify Factories

• Use “dynamic threshold” to intensify the areas with Use “dynamic threshold” to intensify the areas with high values, compared with their surroundings.high values, compared with their surroundings.

Intensity of pixels in the image

Dynamic threshold

Step I – Identify FactoriesStep I – Identify Factories

Locate the centers of these areasLocate the centers of these areas

Step I – Identify FactoriesStep I – Identify Factories

Expand each center to the whole factory areaExpand each center to the whole factory area

Step I – Identify FactoriesStep I – Identify FactoriesUse the “find connected components” functionUse the “find connected components” function to differentiate between factories. to differentiate between factories.

e3 transcription factorye3 transcription factory

RNA Pol IImRNALacI

e3 transcription factorye3 transcription factorymRNA molecules surrounding the gene

RNA pol II and mRNA molecules surrounding the gene

Step II – Calculate CorrelationStep II – Calculate Correlation

• Normalization of the U1 and U2 images.Normalization of the U1 and U2 images.

• Correction of pixel shift.Correction of pixel shift.

Step II – Calculate CorrelationStep II – Calculate Correlation

• We tried several methods to calculate correlation:We tried several methods to calculate correlation:

Pearson coefficientPearson coefficient

average of U1 / average of U2average of U1 / average of U2

curve fit (aX + b)curve fit (aX + b)

Current resultsCurrent results

U1 / U2

Nor

mal

ized

cou

nt

Nor

mal

ized

cou

nt

U1 / U2

Step III – classifying factoriesStep III – classifying factories

• The best method to divide the factories into twoThe best method to divide the factories into two

distinct groups, is ….distinct groups, is ….

• Consensus correlation similar to that of e3 gene Consensus correlation similar to that of e3 gene

high probability of undergoing splicing.high probability of undergoing splicing.

• Different / No consensus correlation Different / No consensus correlation

low probability of undergoing splicing.low probability of undergoing splicing.

The final outputThe final output

• Using color gradient to color factories according to Using color gradient to color factories according to the probability of undergoing splicing.the probability of undergoing splicing.

Biological conclusionsBiological conclusions• A few hundreds of transcription factories in each A few hundreds of transcription factories in each

nucleus, as mentioned in articles from recent years.nucleus, as mentioned in articles from recent years.

? Do factories tend to gather, or do they operate? Do factories tend to gather, or do they operate

throughout the nucleus.throughout the nucleus.

? Do active factories concentrate in the center? Do active factories concentrate in the center

of the nucleus.of the nucleus.

What’s nextWhat’s next??

• Improve factory identification (more automatically).Improve factory identification (more automatically).

• Displaying each factory as an individual objectDisplaying each factory as an individual object

in IMARIS.in IMARIS.

• Analyze more images of e1 and e3 genes, to findAnalyze more images of e1 and e3 genes, to find

the differences between their factories.the differences between their factories.

• Check the correlation of U1 and U4 factors.Check the correlation of U1 and U4 factors.

ThanksThanks

Dr. Yaron Shav-TalDr. Yaron Shav-Tal Mr. Yehuda BrodyMr. Yehuda Brody

http://io9.com/photogallery/scienceart2009/1008301614